In the absence of cAMP, cAMP-dependent protein kinase A (PKA) exists as a heterotetrameric holoenzyme complex composed of two catalytic (C) subunit monomers in complex with a homodimer of one of four regulatory (R) subunit isoforms (RI1, RII1, RI2, or RII2). In response to multiple stimuli, adenylyl cyclase activation produces cAMP which binds to the R subunit dimer causing a conformational change that leads to the release and activation of the catalytic subunit monomers which can phosphorylate target substrates to produce multiple outcomes.1 PKA is ubiquitously expressed in every mammalian cell and is critical for host-pathogen response2, memory3, and energy metabolism.4 PKA is also associated with many diseases including malignancies.5-7 One significant feature of PKA, the N-myristoylation of the C subunit, remains largely uncharacterized. N- myristoylation is the irreversible covalent attachment of myristic acid onto the N-terminal glycine of proteins which typically occurs co-translationally.8,9 Although PKA was the first myristoylated protein identified,10 very little information about the role of this modification has been revealed. N- myristoylation was shown to confer stability to the C subunit,11,12 and it increases the affinity of the holoenzyme complex with the RII1 regulatory subunit isoform for membranes.13 Fluorescence anisotropy experiments also show that the N-terminal region of the C subunit in the RII holoenzyme is much more flexible and therefore able to interact better with membranes compared to the RI holoenzyme. The catalytic subunit alone or in a complex with RI1 appears to bury the myristic acid within the protein making it unlikely to associate with membranes, but these features increase the stability of the C subunit and may affect the RI1 holoenzyme structure.11,13 We hypothesize that myristoylation causes isoform specific changes in PKA structure which, for the RI1 holoenzyme, will cause the protein to be more compact and stable. These changes will aid in achieving our goal to obtain one or more crystal structures of RI1 holoenzyme complexes with truncated, dimeric versions of PKA, truncated tetramers, or even the entire holoenzyme complex. Additionally, with engineered cysteines, we will fluorescently label PKA with fluorescein maleimide to understand protein dynamics using fluorescence polarization. We hypothesize that these techniques will reveal new and important isoform specific dynamics of different PKA holoenzymes that may be important for their function. Finally, we hypothesize that the unique N-terminus of PKA is important in enzyme dynamics, catalysis, and stability. Using site-directed mutagenesis, we plan to test the role of the N-terminus in the biochemistry of the catalytic subunit. The newly identified structures, dynamics, and interactions of multiple PKA holoenzymes may shape the role of PKA in many biological processes.